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Background <p><i>Ralstonia pseudosolanacearum</i> causes bacterial wilt in many crops and survives in dormant states like viable but non-culturable (VBNC), which evade standard detection. While resuscitatable VBNCs are considered pathogenic, non-resuscitatable ones are often assumed harmless. Here, we induce type-I (resuscitatable) and type-II (non-resuscitable) VBNC states via cold (4&#xa0;°C) and UV-C stress in two strains (DIBER-115, DIBER-118), differentiate them using viability, metabolic and resuscitation assays, and test whether type-II VBNCs retain the ability to cause disease in tomato.</p> Methods <p><i>Ralstonia pseudosolanacearum</i> strains DIBER-115 and DIBER-118 were subjected to psychrophilic (4 °C) and UV-C (200–1000 mJ/cm²) stresses. VBNCs were classified as type-I (resuscitating with catalase or extended incubation) and type-II (nonresuscitating but metabolically active, as detected by RT-qPCR). Viability was assessed by SYTO9/PI fluorescence microscopy, and pathogenicity was tested in tomato plants.</p> Results <p>Stochastic type-I VBNCs were detected in untreated controls, whereas stress-induced VBNCs increased by 6–8 Log CFU/ml under cold and UV-C exposure. At 4 °C, viable cells (SYTO9- positive) predominated with &lt; 10% PI-positive cells, confirming persistence during prolonged cold stress. UV-C at 200 mJ/cm² induced type-I VBNCs with pathogenicity (disease incidence, DI: 33.3–40%). At 1000 mJ/cm², a fraction of microcosms survived as type-II VBNCs (DIBER115: 0.38%, DIBER118: 8.50%), which expressed 16 S rRNA but failed to resuscitate. These type-II VBNCs retained pathogenicity despite loss of culturability.</p> Conclusion <p>Our findings support two hypotheses: (i) type-I and type-II VBNCs are distinct phenotypes with different biochemical mechanisms, and (ii) type-II VBNCs can remain pathogenic despite non- resuscitability. The underestimated role of type-II VBNCs in the survival of the <i>Ralstonia solanacearum</i> species complex underscores the importance of combining resuscitation assays with RT-qPCR for accurate detection and highlights their significance for bacterial wilt management.</p>

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Unravelling the dual nature of Ralstonia pseudosolanacearum: pathogenicity of type-II viable but nonculturable cells induced by cold and UV-C stress

  • 期刊论文

  • 发表日期：

    2026年3月26日

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  • Neha Faridi,
  • Shalini Bhatt,
  • Merwyn Samuel,
  • Ankur Agarwal,
  • Veena Pande,
  • Mohammad Tahir Siddiqui

摘要

Background

Ralstonia pseudosolanacearum causes bacterial wilt in many crops and survives in dormant states like viable but non-culturable (VBNC), which evade standard detection. While resuscitatable VBNCs are considered pathogenic, non-resuscitatable ones are often assumed harmless. Here, we induce type-I (resuscitatable) and type-II (non-resuscitable) VBNC states via cold (4 °C) and UV-C stress in two strains (DIBER-115, DIBER-118), differentiate them using viability, metabolic and resuscitation assays, and test whether type-II VBNCs retain the ability to cause disease in tomato.

Methods

Ralstonia pseudosolanacearum strains DIBER-115 and DIBER-118 were subjected to psychrophilic (4 °C) and UV-C (200–1000 mJ/cm²) stresses. VBNCs were classified as type-I (resuscitating with catalase or extended incubation) and type-II (nonresuscitating but metabolically active, as detected by RT-qPCR). Viability was assessed by SYTO9/PI fluorescence microscopy, and pathogenicity was tested in tomato plants.

Results

Stochastic type-I VBNCs were detected in untreated controls, whereas stress-induced VBNCs increased by 6–8 Log CFU/ml under cold and UV-C exposure. At 4 °C, viable cells (SYTO9- positive) predominated with < 10% PI-positive cells, confirming persistence during prolonged cold stress. UV-C at 200 mJ/cm² induced type-I VBNCs with pathogenicity (disease incidence, DI: 33.3–40%). At 1000 mJ/cm², a fraction of microcosms survived as type-II VBNCs (DIBER115: 0.38%, DIBER118: 8.50%), which expressed 16 S rRNA but failed to resuscitate. These type-II VBNCs retained pathogenicity despite loss of culturability.

Conclusion

Our findings support two hypotheses: (i) type-I and type-II VBNCs are distinct phenotypes with different biochemical mechanisms, and (ii) type-II VBNCs can remain pathogenic despite non- resuscitability. The underestimated role of type-II VBNCs in the survival of the Ralstonia solanacearum species complex underscores the importance of combining resuscitation assays with RT-qPCR for accurate detection and highlights their significance for bacterial wilt management.